THE SELENIUM RECYCLING ENZYME SELENOCYSTEINE LYASE: REGULATION AND PHYSIOLOGICAL ROLE IN GLUCOSE AND LIPID METABOLISM A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI‘I AT MĀNOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN CELL AND MOLECULAR BIOLOGY MAY 2012 BY LUCIA ANDREIA SEALE Dissertation Committee: Marla J. Berry, Chairperson Robert A. Nichols Jun Pane‘e P. Reed Larsen E. Gordon Grau To my beloved parents Wilson & Madalena To my dear love André ii ACKNOWLEDGEMENTS A Ph.D. is a journey in a rough sea. As in all memorable expeditions, there is more to learn while traveling towards the final destination than at the destination itself. In several ways, it is the path and how you face the unexpected obstacles in this path that makes you a Doctor. The Ph.D. expedition does not allow an easy ride. While you travel through the waves and storms of your topic, figuring out a lot on and about yourself, you also encounter along the way people that make your stressful journey happier, nicer, safer, funnier and – why not – more challenging. They provided the emotional background that is not described in a scientific document. In my Ph.D. journey, these are the people about whom I’ll talk years ahead to friends, to whom I will silently smile when the nostalgic waves of the Ph.D. path break in the shores of my life. They are the unforgettable people that allowed me to arrive safe and sound in Doctorland. First and foremost, my greatest acknowledgement and gratitude goes to Dr. Marla Berry. Her incredible support and mentoring skills throughout several moments of my career is just unprecedented and truly amazing. On the occasion I still regard as one of the most important moments of my life, I was leaving the cold room in a cold Harvard laboratory in a freezing Boston winter, and Dr. Berry warmly offered me to join her science team. From that day on, she consistently fostered my curiosity towards the intricacies of the selenium field in a very positive, inspirational and rewarding way. As if all of this incredible support wasn't enough, she was pivotal in bringing me to the place on Earth my heart calls home. I wish to especially thank my Ph.D. Committee: Dr. Robert Nichols, Dr. Jun Pane'e, Dr. P. Reed Larsen and Dr. E. Gordon Grau. Their intense questioning and constructive arguments set the tone for this entire work. They brought perspectives that threw me out of the comfort zone in name of a better scientific outcome. They have been astonishing for reminding me through their examples of accomplishment, scientific productivity and humbleness that the Ph.D. journey may end, but the learning does not. I would like to extend a very special mahalo to Mrs. Ann Hashimoto. Her organization, diligence, care, talent and incredible commitment for the experimental mice led to the success of the results presented herein. In addition, she provided a warm and iii healthy environment for the daily work with her smiles and pleasant talks. Mahalo also to my labmates Ms. Christy L. Gilman and Mr. Ali Seyedali, for assisting me in several experiments, providing good insights, patiently enduring my terrible daily jokes, and being unforgettable "skilled sailors in the rough seas" of science. In addition, I would like to thank Mr. Arjun Raman and Dr. Frederick Bellinger for their collaboration and insightful selenium discussions beyond the scope of the available literature. I would also like to especially thank my outside collaborators, who made specific contributions that helped turn this project into reality: Dr. Ann-Marie Zavacki, Dr. Alessandro Marsili, Dr. Nicholas V. Ralston, Dr. Laura Raymond, Dr. Ashok Balasubramanyam, Dr. Guylaine Poisson and Mr. Mahdi Belcaid. I thank the colleagues at the John A. Burns School of Medicine, all of whom at some point of my Ph.D. journey helped with their expertise and/or positive interaction and/or made me smile or reflect upon several scientific issues in an aloha atmosphere. Among them are: Mrs. Miyoko Bellinger, Ms. Mindy McDermott, Mr. Jason K. Higa, Ms. Xiaosha Pang, Dr. Cedomir Todorovic, Dr. Matthew Pitts, Dr. Suguru Kurokawa, Ms. Ashley Kong, Dr. Mariana Gerschenson, Dr. Suguru Kurokawa, Mrs. Elizabeth Nguyen- Wu, Ms. Kerri Chock, Ms. Rachel Rueli, Mrs. Fukun Hoffmann. I also thank the incredible administrative staff of the Department of Cell and Molecular Biology at the John A. Burns School of Medicine that clarified several of the bureaucratic steps required for a Ph.D. I thank also the entities that financially supported my projects: the National Institutes of Health (R01 DK47320, R56 DK47320 and G12 MD007601/RR003061), the Graduate Student Organization with its Travel Award, and the Department of Cell and Molecular Biology Graduate Program with its Graduate Assistantship. Lastly, but far from least, a special thank you to my online and offline friends. During the Ph.D. journey, when the seas were really rough, your relaxing words posted on twitter or facebook walls, in blog comments, or during countless happy hours pointed me to esteemed perspectives required for a safe journey. None of this work would be possible without the passionate support I get from my family, especially my parents, and from my lovely husband André, who has always been there for me, providing constructive advice, and whose tireless encouragement was a daily and cheerful reminder that the PhD journey, although rough, one day reaches the academic hub from where many other new and exciting journeys may depart. iv ABSTRACT Selenium (Se) is an essential trace element used for biosynthesis of selenoproteins, acquired either through the diet or cellular recycling mechanisms involving the enzyme selenocysteine lyase (Scly). This dissertation characterizes the Se- dependent regulation of Scly gene expression in in vitro and in vivo models. Scly mRNA negatively correlates with Se levels in HepG2 cells, liver and brain tissues, but not in Ht22 cells. Scly is also downregulated in the liver of the Selenoprotein P (Sepp1) knockout (KO) mouse, a model lacking the Se transporter protein with elevated hepatic Se content. Additionally, this dissertation reports the development and characterization of a Scly KO mouse. This mouse model presents diminished Se content and increased expression of selenoprotein mRNAs in the liver compared with wild type mice. Scly KO mice maintain circulating Se levels and most selenoprotein mRNA levels in Se target tissues, such as brain and testis. Disruption of the Scly mechanism also affects hepatic energy metabolism. Scly KO mice raised on a Se adequate diet exhibit hyperinsulinemia and mild hepatic steatosis, with attenuated insulin signaling as measured by Akt phosphorylation and increased oxidative stress, without changes in phosphorylation or protein levels of AMP-activated Protein Kinase alpha, a regulator of cholesterol biosynthesis. On Se restriction, Scly KO mice develop characteristics of metabolic syndrome, such as obesity, fatty liver, hypercholesterolemia and insulin resistance. Moreover, hepatic glutathione peroxidase 1 and circulating Sepp1 levels are also diminished, but mRNA levels of Acetyl-CoA carboxylase are increased. When exposed to a high fat, Se adequate diet, Scly KO mice become more vulnerable to obesity. The findings of this dissertation unveil a strong connection between Se recycling and glucose and lipid metabolism, and demonstrate for the first time in an animal model the unique physiological role of the Se recycling pathway in lipid and glucose metabolism. v TABLE OF CONTENTS Page Acknowledgements ................................................................................................. iii Abstract ................................................................................................................... v Table of Contents .................................................................................................... vi List of Figures .......................................................................................................... xi List of Tables ........................................................................................................... xiii List of Abbreviations ................................................................................................ xiv Chapter 1: Introduction 1.1. History of Selenium and Selenium Research ....................................... 01 1.2. Dietary Selenium .................................................................................. 02 1.2.1. Se intake ................................................................................ 02 1.2.2. Chemical forms ...................................................................... 03 1.3. Selenocysteine Incorporation Mechanism and Selenoprotein Biosynthesis................................................................................................. 05 1.4. Selenoproteins ..................................................................................... 07 1.4.1. Serum selenoproteins: GPx3 and Sepp1 .............................. 09 1.4.2. Hierarchy of selenoprotein production .................................. 11 1.4.3. Selenoproteins and human health ........................................ 12 1.5. Selenocysteine Lyase .......................................................................... 15 1.6. Physiological Role of Scly .................................................................... 17 1.7. Research Objectives ...........................................................................